JPH04273826A - Anti-rs viral agent - Google Patents

Abstract

PURPOSE:To obtain preventive and remedy for human and animal infectious diseases caused by infection of RS virus (Respiratory Syncytial virus). CONSTITUTION:An anti-RS viral agent containing N-[N-(N-benzyloxycarbonyl-L- phenylalanyl)-L-leucyl] glycine as an active constituent. The viral agent has low toxicity to mammal and has excellent selective anti-RS virus activity.

Description

[Detailed description of the invention]

0001

[Field of Industrial Application] The present invention relates to an antiviral agent, and more specifically to an antiviral agent for respiratory syncytial virus (RS virus).
The present invention relates to a drug for the prevention and treatment of infectious diseases in humans and animals caused by infection with syncytial viruses.

0002

[Prior Art] RS virus (R
espiratory Syncytial vi
Rus) is known as the most important and dangerous pathogenic virus that frequently infects young children and causes severe respiratory symptoms. Several types of antiviral agents are known, including nucleic acid compounds such as the antiherpes drug acyclovir, amantadine, and its derivatives, but all of them have low selectivity against viruses, are highly toxic to mammals, and have limited therapeutic efficacy. Satisfactory results regarding the relationship between toxicity and toxicity have not yet been obtained. However, in recent years, the method of virus infection into host cells has been studied at the molecular level, and the amino acid sequence of the fusion peptide, which acts when the virus adsorbs to the host cell surface and injects the viral genome into the host cell, has been elucidated. It started to be done. Along with this, research has begun to synthesize compounds similar to viral fusion peptides to selectively exert an inhibitory effect on viruses. For example, Chopin et al. reported that a compound similar to the compound of the present invention, N-[N-(N-benzyloxycarbonyl-D-phenylalanyl)-L-phenylalanyl]glycine, is a compound similar to the compound of the present invention.
xovirus family, myxovirus
has been reported to have inhibitory activity against viruses of the U.S. family (Purnell W. Choppin).
etc. al Virology Vol. 105
205-222 (1980)).

[0003] Although Chopin et al. did not report the inhibitory activity of these compounds against RS virus, the present inventors found that during the research process of the present invention, the compounds prepared by Chopin et al. It was revealed that it had no activity at all.

0004

[Problems to be Solved by the Invention] Production of a vaccine against RS virus, which is an attenuated version of RS virus, has been discontinued due to doubts about its effectiveness. Also RS
Ribavirin, a nucleic acid compound, has been shown to be effective against viruses, but ribavirin is also highly toxic and there is still no drug that has clinically sufficient therapeutic effects against RSV. Nosocomial infections have become a major problem for clinicians. Particularly for infants, the toxicity of the drugs used is an important issue, and there has been a desire to develop anti-RS virus drugs that have sufficient preventive and therapeutic effects and are low in toxicity.

[0005]

[Means for Solving the Problems] In order to solve the conventional problems, the present inventors have discovered that RS virus adsorbs to the surface of host cells.
As a result of synthesizing many compounds with amino acid sequences similar to fusion peptides that act when injecting the viral genome into host cells and conducting intensive research on compounds with selective anti-RS virus activity, we found that N-[N-( The present invention was completed by discovering that N-benzyloxycarbonyl-L-phenylalanyl-L-leucyl]glycine has excellent anti-RS virus activity and that its toxicity to mammals is extremely low.

The present invention relates to N-[N-(N-benzyloxycarbonyl-L-phenylalanyl)-L-leucyl]
The present invention relates to an anti-RS virus agent containing glycine as an active ingredient. More specifically, the present invention relates to a drug for preventing and treating infectious diseases in humans and animals caused by infection with RS virus. The term RS virus infection as used herein refers to diseases such as pharyngitis, bronchitis, and pneumonia caused by RS virus infection, as well as complications caused by RS virus and bacterial infection. It is also known that RS virus causes infections in mammals other than humans, and the anti-RS virus agent of the present invention is also effective against infections in these animals.

The active ingredient N-[N-(N-benzyloxycarbonyl-L-phenylalanyl)-L-leucyl]glycine of the present invention is L-phenylalanine whose amino group is protected with an N-benzyloxycarbonyl group. ni, L-
It is a tripeptide consisting of three amino acids in which leucine and then glycine are peptide-bonded, and it is produced by a known method for producing peptides.

[0008] The drug for preventing and treating RS virus infection according to the present invention can be administered orally or parenterally (for example, intravenously, intradermally, rectally, by spray inhalation, or intranasally). In this case, a dosage form suitable for each administration method can be prepared. Depending on the use, such drugs may be tablets, capsules, granules, powders, fine granules, pills, troches, sublinguals, suppositories, ointments, injections, emulsions, suspensions, sprays, It can be prepared in any formulation form such as nasal spray or syrup. The preparation of these formulations includes non-toxic solvents, excipients, binders, lubricants, disintegrants, preservatives, tonicity agents, stabilizers, dispersants, antioxidants, colorants, flavoring agents, It can be formulated by known methods using additives such as buffers. The content of the compound of the present invention in the preparation varies depending on its dosage form, but it is generally desirable to contain the compound at a concentration of 0.1 to 100% by weight. The dosage of the preparation can vary widely depending on the type of mammal including humans, the severity of symptoms, diagnosis by a doctor, etc., but can generally be 0.01 to 300 mg/kg per day. This dosage can be administered once a day or in divided doses.

[0009]

[Examples] Examples of the present invention are shown below, but the present invention is not limited to the examples shown below. (Hereinafter, N-tert-butoxycarbonyl group is referred to as Boc
, N-benzyloxycarbonyl group is abbreviated as Z, L-phenylalanine as L-Phe, L-leucine as L-Leu, and glycine as Gly. )

(Example 1) Boc-L-Leu-G
Synthesis of N-[N-(N-benzyloxycarbonyl-L-phenylalanyl)-L-leucyl]glycine (Z-L-Phe-L-Leu-Gly) using lyOEt.

Boc-L-Leu (N-te
rt-butoxycarbonyl-L-leucine) (462m
g, 2 mmol), ethoxyglycinate hydrochloride (hereinafter referred to as
(abbreviated as GlyOEt・HCl) (279mg, 2m
mol), triethylamine (0.5 ml), 1-hydroxybenzotriazole (hereinafter abbreviated as HOBt) (270 mg, 2 mmol), dicyclohexylcarbodiimide (hereinafter abbreviated as DCC) (412 mg,
2 mmol) was reacted in 10 ml of dichloromethane at room temperature for 8 hours to quantitatively obtain Boc-L-Leu-GlyOEt. Next, Boc-L-Leu-GlyOEt(
Z-L-Phe (299 mg, 1 mmol) was treated with 25% HBr-acetic acid for 1 hour at room temperature.
mol), triethylamine (0.5ml), HOBt
(135 mg, 1 mmol), DCC (206 mg, 1
mmol) in 10 ml of dichloromethane at room temperature for 8 hours to produce Z-L-Phe-L-Leu-GlyOE.
t was obtained quantitatively.

Below, Z-L-Phe-L-Leu-Gl
The physical property values of yOEt are shown.

mp: 147-149°C, IR (CHCl
3): 3450, 1742, 1725, 1672 cm-
1

NMR (CHCl3) δ: 0.89 (6
H, d, J=5Hz, (CH3)2CH), 1.23(
3H, t, J=6Hz, OCH2CH3), 1.33-
1.85 (3H, m, CH2CH(CH3)2), 3.
03 (2H, d, J=7Hz, CCH2Ph), 3.8
7 (2H, brs, NCH2CO), 4.37 (2H,
q, J=6Hz, OCH2CH3), 4.18-4.7
2 (2H, m, NCHCO x 2), 5.02 (2H, s
, OCH2Ph) 5.50-5.66 (1H, brs,
NH), 6.65-6.82 (1H, brs, NH),
7.15 (5H, s, C6H5), 7.23 (5H, s
, C6H5), 7.96 (1H, s, NH)

[α]D22=-27.3° (c=0.3
, MeOH), high-resolution mass spectrum: C27H35
N3O6 (M+) Theoretical value: 497.2524, Actual value:
497.2538

Next, Z-L-Phe-L-Leu-G
lyOEt (248.5 mg, 0.5 mmol) in methanol (4 ml) and 2 ml of 5% NaOH aqueous solution,
Hydrolysis was carried out at room temperature for 1.5 hours. After methanol is distilled off under reduced pressure, the aqueous solution is acidified with citric acid to precipitate Z-L-Phe.
-L-Leu-Gly crystals were collected by filtration. Moreover, the yield was 185 mg (79%).

Hereinafter, Z-L-Phe-L-Leu-Gl
Indicates the physical property value of y.

mp: 154-155°C, IR (CHCl
3): 3350, 1720, 1670 cm-1

001
9] NMR (CHCl3) δ: 0.86 (6H, d, J
=5Hz, (CH3)2CH), 1.36-1.75(
3H, m, CH2CH(CH3)2), 2.92(2H
, d, J=5Hz, CH2Ph), 3.97(2H, b
rs, NCH2CO), 4.37-4.79 (2H, m
, NCHCO×2), 5.05 (2H,s, OCH2P
h), 5.63-5.93 (1H, brs, NH), 6
．． 87-7.76 (12H, m, C6H5×2, NH×
2)

[α]D22=-33° (c=0.2
, MeOH), high-resolution mass spectrum: C25H31
N3O6 (M+) Theoretical value: 469.2211, Actual value:
469.2235

(Example 2) Z-L-Leu-Gly
N-[N-(N-benzyloxycarbonyl-L-phenylalanyl)-L-leucyl]glycine (Z-L-Phe-L-Leu-Gly) was synthesized using OEt.

[0022] Z-L-Leu (1.115g, 4.2m
mol), GlyOEt・HCl (586 mg, 4.2
mmol), DCC (867 mg, 4.2 mmol),
HOBt (568 mg, 4.2 mmol) and triethylamine (3 ml) were reacted in dichloromethane (50 ml) at room temperature for 8 hours to quantitatively obtain Z-L-Leu-GlyOEt. Z-L-Leu-GlyOEt (350m
g, 1 mmol) was dissolved in 10 ml of methanol, deprotected by catalytic reduction with palladium carbon (50 mg), and then Z-L-Phe (299 mg, 1 mmol) was dissolved in 10 ml of methanol.
), HOBt (135 mg, 1 mmol), triethylamine (0.1 ml), DCC (206 mg, 1 mmol)
l) in dichloromethane (10 ml) to form Z
-L-Phe-L-Leu-GlyOEt was obtained. Further, this was treated in the same manner as in Example 1 to obtain Z-L-Phe-L.
-Leu-Gly was obtained.

(Experimental Example 1) The anti-RS virus activity of Z-L-Phe-L-Leu-Gly according to the present invention obtained in Examples 1 and 2 above was measured.

[0024] In order to measure anti-RS virus activity, He
Tests were conducted with RSV-Long strain using La cells. 2
HeLa cells were cultured in a 4-well microplate at 35°C for 2 days, and when they became a monolayer, the growth medium (GM) was discarded and the final concentrations were 200, 40, 8, 1.6, 0.3, and 0 μg. /ml with cell maintenance solution (MS).
The drug solution diluted with 7% methyl cellulose solution was
Added ml at a time. In addition, 50 plaque forming units are placed in each hole.
, PFU) was added and cultured at 35°C for 4 days in a 5% carbon dioxide incubator.The presence or absence of plaque in each well was observed under a microscope, and the EC50
(Effective Concetration)
I asked for Z-L-Phe-L-Leu-Gl of the present invention
The EC50 of y was 2.7 μg/ml or less, and it showed a strong inhibitory effect on RS virus.

(Experimental Example 2) A cytotoxicity test was conducted on Z-L-Phe-L-Leu-Gly according to the present invention obtained in Examples 1 and 2 above.

[0026] HeLa cells were cultured in a 24-well microplate at 35°C for 2 days, and when they became a monolayer, the growth medium (GM) was replaced with a drug solution, and morphological changes were observed using a microscope every day to determine whether the drug The toxicity to cells was investigated. Cell viability was measured on the 2nd and 7th day after drug addition.
ty) is reduced to 50% of the control (C
C50: cytotoxic concentration
ion) was calculated.

Z-L-Phe-L-Leu-G of the present invention
The CC50 of ly is 200 μg/ml or more, and it is clear that it is an extremely safe compound that does not show any inhibitory effect on normal cells.

The results of Experimental Examples 1 and 2 are shown in Table 1 below.

[Table 1]

As is clear from the results of Experimental Examples 1 and 2 above, L-Ph of Z-L-Phe-L-Leu-Gly
No activity was observed when e was changed to D-Phe, indicating that optical activity is important for the expression of inhibitory activity. Also, Z-D-Phe-D-Phe-G of Chopin etc.
ly and Z-D-Phe-L-Phe-Gly showed no inhibitory activity against RS virus. From these results, it is clear that RS virus is a highly specific virus, unlike other viruses, and that the compound according to the present invention specifically inhibits RS virus.

(Experimental Example 3) A toxicity test was conducted on Z-L-Phe-L-Leu-Gly according to the present invention obtained in Examples 1 and 2 above using mice.

Z-L-Phe-L-Leu-Gly80
Dissolve mg in 1 ml of ethanol, dilute 10 times with physiological saline to prepare a sample solution, and give a dose of 40 mg/kg/
The drug was administered intravenously to 5 mice once a day for 3 consecutive days, but there were no deaths and no abnormalities that appeared to be caused by drug administration were observed. In addition, a similarly prepared sample solution was administered intraperitoneally to 5 mice at a dose of 40 mg/kg/day once a day for 3 consecutive days, but there were no deaths and no abnormalities that appeared to be due to drug administration. I was not able to admit. Further Z-L-Phe-L-Leu-Gly40
After dissolving 0 mg in 1 ml of ethanol, dilute with physiological saline for 10 min.
A twice diluted sample solution was orally administered to five mice at a dose of 2 g/kg, but there were no deaths and no abnormalities that appeared to be caused by drug administration were observed. From these results, it is clear that Z-L-Phe-L-Leu-Gly of the present invention is an extremely safe compound for mammals.

(Example 3) An injection was prepared using Z-L-Phe-L-Leu-Gly according to the present invention obtained in Examples 1 and 2 above.

Z-L-Phe-L-Leu-Gly50
mg was dissolved in 1 ml of ethanol, diluted by adding 9 ml of sterilized physiological saline, filtered through a membrane filter (pore size: 0.45 μm), and sealed in a sterilized ampoule bottle to prepare an injection ampoule.

(Example 4) A spray was prepared using Z-L-Phe-L-Leu-Gly according to the present invention obtained in Examples 1 and 2 above.

Z-L-Phe-L-Leu-Gly25
mg was dissolved in 0.5 ml of ethanol, diluted by adding 9.5 ml of physiological saline, filtered through a membrane filter (pore size: 0.45 μm), and sealed in a sterilized vial to prepare a formulation for a spray inhaler. did.

(Example 5) A 25 mg capsule was prepared using Z-L-Phe-L-Leu-Gly according to the present invention obtained in Examples 1 and 2 above.

Z-L-Phe-L-Leu-Gly25
After grinding mg well, 50 mg of lactose, 23 mg of starch
mg and 2 mg of magnesium stearate were added thereto, mixed thoroughly, and then filled into capsules to prepare 25 mg capsules.

[0038]

[Efficacy] The anti-RS virus agent of the present invention has extremely low toxicity to mammals and exhibits a specific viral activity inhibitory effect on RS virus. This makes it possible to provide an extremely effective anti-RS virus agent for RS virus infection, which has not been seen before.

Claims (1)

[Claims] 1. An anti-RS virus agent comprising N-[N-(N-benzyloxycarbonyl-L-phenylalanyl)-L-leucyl]glycine as an active ingredient.